Reduction of entropic barrier of polyelectrolyte molecules in a nanopore device with agarose gel

ABSTRACT

A mechanism is provided for reducing entropy of a polyelectrolyte before the polyelectrolyte moves through a nanopore. A free-standing membrane has the nanopore formed through the membrane. An agarose gel is formed onto either and/or both sides of the nanopore in the membrane. The agarose gel is a porous material. The polyelectrolyte is uncoiled by driving the polyelectrolyte through the porous material of the agarose gel via an electric field. Driving the polyelectrolyte, having been uncoiled and linearized by the agarose gel, into the nanopore is for sequencing.

DOMESTIC PRIORITY

This application claims priority to Provisional Application No.61/976,562, filed on Apr. 8, 2014, which is herein incorporated byreference in its entirety.

BACKGROUND

The present invention relates to nanopore/nanochannel devices, and morespecifically, to reducing the entropy of molecules via agarose gel innanopore/nanochannel devices.

Nanopore sequencing is a method for determining the order in whichnucleotides occur on a strand of deoxyribonucleic acid (DNA). A nanopore(also referred to a pore, nanochannel, hole, etc.) can be a small holein the order of several nanometers in internal diameter. The theorybehind nanopore sequencing is about what occurs when the nanopore issubmerged in a conducting fluid and an electric potential (voltage) isapplied across the nanopore. Under these conditions, a slight electriccurrent due to conduction of ions through the nanopore can be measured,and the amount of current is very sensitive to the size and shape of thenanopore. If single bases or strands of DNA pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore. Other electrical oroptical sensors can also be positioned around the nanopore so that DNAbases can be differentiated while the DNA passes through the nanopore.

The DNA can be driven through the nanopore by using various methods, sothat the DNA might eventually pass through the nanopore. The scale ofthe nanopore can have the effect that the DNA may be forced through thehole as a long string, one base at a time, like thread through the eyeof a needle. Recently, there has been growing interest in applyingnanopores as sensors for rapid analysis of biomolecules such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, etc.Special emphasis has been given to applications of nanopores for DNAsequencing, as this technology holds the promise to reduce the cost ofsequencing below $1000/human genome.

SUMMARY

According to an embodiment, a method for reducing entropy of apolyelectrolyte before the polyelectrolyte moves through a nanopore isprovided. The method includes providing a free-standing membrane havingthe nanopore formed through the membrane, and forming an agarose gelonto at least one of either or both sides of the nanopore in themembrane. The agarose gel is a porous material. The method includeuncoiling the polyelectrolyte by driving the polyelectrolyte through theporous material of the agarose gel via an electric field, and drivingthe polyelectrolyte, having been uncoiled and linearized by the agarosegel, into the nanopore for sequencing.

According to an embodiment, a system for reducing entropy of apolyelectrolyte before the polyelectrolyte moves through a nanopore isprovided. The system includes a free-standing membrane having thenanopore formed through the membrane, a top fluidic reservoir on oneside of the membrane and a bottom fluidic reservoir on an opposing sideof the membrane, and an agarose gel formed onto at least one of eitheror both sides of the nanopore in the membrane. The agarose gel is aporous material. An electric field generated by a voltage source drivesthe polyelectrolyte through the porous material of the agarose gel touncoil the polyelectrolyte. Driving the polyelectrolyte, having beenuncoiled and linearized by the agarose gel, into the nanopore is forsequencing.

Additional features and advantages are realized through the techniquesof the present invention. Other embodiments and aspects of the inventionare described in detail herein and are considered a part of the claimedinvention. For a better understanding of the invention with theadvantages and the features, refer to the description and to thedrawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The subject matter which is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The forgoing and other features, and advantages ofthe invention are apparent from the following detailed description takenin conjunction with the accompanying drawings in which:

FIG. 1A a process flow to fabricate a free-standing membrane with ananometer pore according to an embodiment.

FIG. 1B illustrates forming a cavity in the backside of the membraneaccording to an embodiment.

FIG. 1C illustrates forming the nanometer pore in the membrane accordingto an embodiment

FIG. 2A illustrates a cross-sectional view of a fluidic chamber with atop fluidic reservoir and a bottom fluidic reservoir according to anembodiment.

FIG. 2B illustrates agarose gel formed on both sides of the nanoporeaccording to an embodiment.

FIG. 2C illustrates that the polyelectrolyte is injected into the topfluidic reservoir as a coil according to an embodiment.

FIG. 2D illustrates that the polyelectrolyte is linearized and uncoiledby passing through the agarose before entering into the nanoporeaccording to an embodiment.

FIG. 2E illustrates the full length of the linearized and uncoiledpolyelectrolyte within the agarose gel and through the nanoporeaccording to an embodiment.

FIG. 3 illustrates multilayers (gradient) of agarose gels with differentconcentrations corresponding to different pore sizes according to anembodiment.

FIG. 4 illustrates a method for reducing entropy of a polyelectrolytebefore the polyelectrolyte moves through the nanopore according to anembodiment.

FIG. 5 is a block diagram that illustrates an example of a computer(computer test setup) having capabilities, which may be included inand/or combined with embodiments.

DETAILED DESCRIPTION

Embodiments provide a method and nanopore device to reduce the entropicbarrier to nanopores with agarose gel for polyelectrolyte sensing, suchas DNA or RNA sequencing.

The nanopore device (which has holes with a few nanometers in diameter)is a promising technology for next-generation DNA sequencing. Itconsists of a planar substrate with a thin insulating membrane(typically tens of nanometers thick) with a nanometer-sized pore drilledthrough. When the polyelectrolyte (DNA, RNA, peptide, etc.) is loaded inthe conductive liquid on both sides of the membrane, the polyelectrolyteis forced by an external field to translocate through the nanoporeopening. Hence, electrical signals can be recorded to detect themolecules, nucleotides and/or bases of the polyelectrolyte.

To make the nanopore devices reliable for fast sequencing, severalcriteria may need to be satisfied. First, the polyelectrolyte needs tobe linearized, rather than coiled, in the nanopore for reliablesequential reading. Second, for uniform motion control ofpolyelectrolytes which are longer than the membrane thickness, theelectric field (E) needs to be applied uniformly across the wholemembrane and extend into the conductive liquid. Third, a pre-stretchingof the polyelectrolyte before its entry into the nanopore is requiredfor fast translocation without clogging the nanopore.

However, the above challenges may still remain unresolved inconventional nanopore devices that rely only on a thin and single-layermembrane (biological or artificial) and pose very large entropicbarriers to long polyelectrolyte molecules. In the bulk liquid solutionof such devices, where there is no geometrical confinement, the longpolyelectrolyte molecules stay in a coiled state and their entropy isthe largest in this coiled state; in contrast, within the nanoporeregion, the polyelectrolyte molecules experience great confinement andforce themselves to decoil into a linear state, corresponding to verysmall entropy.

Such a huge entropy difference (between the coiled and uncoiled statesfor the polyelectrolyte) causes a significantly high energy barrier forthe long polyelectrolyte to overcome and hence translocate, and the hugeentropy difference greatly lowers the translocation speed of thepolyelectrolyte through the nanopore. Furthermore, an abrupt electricfield change over a very small distance at the nanopore entrancevicinity can drive in the coiled polyelectrolyte into the confinednanopore, and thus cause very long clogging events. Additionally, such alarge entropy change can cause configurational instabilities of thepolyelectrolyte molecules and even drive the polyelectrolyte to coil anddecoil (i.e., uncoil) inside the nanopore. All the above issues can leadto reduced and clogged events and thus severely affect the properdetection of bases and/or nucleotides.

Conventionally, different methods are used to enhance the translocationsof DNA for nanopores, like salt gradient and high voltages. Thosemethods only change the electrical field distribution close to thenanopores entry. Salt gradient structures were used to uncoil the DNAbefore it moved through nanochannels, because those micro-structures canhelp to reduce the entropy barrier to nanochannels. However, suchmicro/nano-patterned structures are very difficult to integrate into thevicinities of the nanopore openings.

Embodiments provide effective methods that utilize agarose gels toimprove the electric field uniformity and also lower the entropicbarrier, all of which helps the polyelectrolyte to move through nanoporein a linearized (straight) state.

Embodiments have various benefits by utilizing the agarose gel asdiscussed herein: (1) the agarose gel provides millimeter-scalenanoconfinement to the polyelectrolyte molecules and pre-stretch thembefore their entry into the nanopores, hence greatly lowering theentropic barrier; (2) the agarose gel also greatly reduces the bulkconductivity and extends the electric fields much longer than (i.e.,uniformly beyond) the membrane itself, which is beneficial to highermolecular capture rate and higher translocation events; and (3) theagarose gel has different porous structures at different concentrationsof agarose, and thus provides easy tuning of the polyelectrolyteconfinement and electric field distribution.

Embodiments integrate agarose gel and the nanopore device together forDNA sequencing. The agarose gel uncoils the DNA before reaching theentry of the nanopore. Then, the nanopore reads the single baseinformation when the single-stranded DNA passes through nanopore. Themethod helps the DNA move through nanopore by reducing the entropicbarrier to nanopore entry.

Now turning to the figures, FIGS. 1A, 1B, and 1C (generally referred toas FIG. 1) illustrate the process flow to fabricate a membrane 100 witha single nanometer pore (i.e., nanopore) according to an embodiment.FIG. 1 shows a cross-sectional view of the membrane 100 which is ananopore device.

In FIG. 1A, substrate 101 is any electrically insulating substrate (suchas silicon formulated to be electrically insulating). Insulating layers102 and 103 are respectively deposited on the bottom and top of thesubstrate 101. The insulating layers 102 and 103 are electricallyinsulating films, such as silicon nitride, silicon dioxide, etc. Theinsulating layer 102 protects the bottom of the substrate 101.Insulating layers 102 and 103 are the etch mask to form a cavity 104 inFIG. 1B. The cavity 104 may be fabricated by standard semiconductorprocesses, like wet etch tetramethylammonium hydroxide (TMAH), potassiumhydroxide (KOH), etc.

In FIG. 1C, a nanopore 105 is formed through the top insulating layer103. The nanopore 105 is a hole that is few nanometers (e.g., 2, 3, 4,5, . . . 8 nm) in diameter, and the nanopore 105 can be fabricated by areactive ion etch method, transmission electron microscopy (TEM), and/orhelium ion microscopy (HIM). The nanopore 105 can have different shapes,such as single conical shape, a double conical shape, and/or cylindricalshape. Also, the nanopore 105 may be a nanochannel in someimplementations.

FIGS. 2A, 2B, 2C, 2D, and 2E (generally referred to as FIG. 2)illustrate a system 200 incorporating the membrane 100 (i.e.,nanodevice) according to an embodiment. FIG. 2 illustrates across-sectional view of the system 200.

FIG. 2A shows a fluidic chamber with a top fluidic reservoir 201 and abottom fluidic reservoir 202. The membrane 200 is sealed to andseparates the top and bottom fluidic reservoirs 201 and 202. Althoughtop and bottom fluidic reservoirs are discussed, it is contemplated thatthe fluidic chamber may be left and right sides.

The top and bottom fluidic reservoirs 201 and 202 may be made of anyelectrically insulating materials, such as Teflon®, acrylic, and soforth. The top reservoir 201 and bottom reservoir 202 will be filledwith a conductive fluid 250.

FIG. 2B shows agarose gel 207 and 208 formed on both sides of thenanopore 105. The agarose gel 207 is formed on the top side of thenanopore 105, and the agarose gel 208 is formed on the bottom side ofthe nanopore 105. For example, the agarose gel 207 may be poured ontothe top side of the insulating layer 103 through the top fluidic chamber201, such that the agarose gel 207 forms on the insulating layer 103 toa desired thickness. An example thickness of the agarose gel 207 may be1 to a few (2-9) millimeters (or even centimeters thick).

The agarose gel 208 may be poured onto the bottom side (backside) of theinsulating layer 103 through the bottom fluidic chamber 202, such thatthe agarose gel 208 forms on the backside of the insulating layer 103 toa desired thickness. An example thickness of the agarose gel 208 may be5 millimeters. Note that the agarose gel is too thick to flow into thenarrow diameter of the nanopore 105.

The conductive fluid 250 also fills the top and bottom fluidic chambers201 and 203. FIG. 2C illustrates that the polyelectrolyte such as a DNAmolecule 213 is injected into the top fluidic reservoir 201. FIG. 2Cshows a voltage source 209 and an ammeter 210 (ampere meter). Theammeter 210 monitors the current change of the nanopore 105 when voltageof the voltage source 209 is applied. Electrodes 211 and 212 arerespectively placed in the conductive fluid 250 of the top and bottomfluidic chambers 201 and 202. The electrodes 211 and 212 may be made ofAg or AgCl. The DNA molecule 213 is pulled through nanopore 105 by theuniform electrical field E (shown by the downward pointing arrow that isactually through the membrane 100 and agarose gels 207, 208 but shownoutside so as not to obscure the figures) between electrodes 211 and 212when voltage is applied by the voltage source 209. Note that the DNAmolecule 213 is in a coil (i.e., a ball) before the DNA molecule 213enters the nanopore 105. A large DNA molecule 213 may have a coil (i.e.,coil space) of 1×10⁻⁶ meters (microns, micrometers, and/or μm) indiameter. While coiled, the DNA molecule 213 has a large entropicbarrier to entering the nanopore 105.

However, the agarose gel 207 provides millimeter-scale nanoconfinementto the DNA molecule 213 and pre-stretches (i.e., uncoils) the DNAmolecule 213 before the DNA molecule 213 enters the nanopore 105, whichgreatly lowers the entropic barrier. The voltage of the voltage source209 generates the electric field (E) that drives the DNA molecule 213into the agarose gel 207 and eventually into (and through) the nanopore105. Yet, before the DNA molecule 213 reaches the entry of the nanopore105, the DNA molecule 213 is uncoiled as the DNA molecule 213 passesthrough the agarose gel 207 as shown in FIG. 2D. FIG. 2D shows that theDNA molecule 213 is linearized before the DNA molecule 213 enters intothe nanopore 105. The DNA molecule 213 keeps the linearized status(i.e., uncoiled) when the DNA molecule 213 stays inside the agarose gel207 and/or agarose gel 208 as shown in FIGS. 2D and 2E. FIG. 2E showsthe full length of the linearized DNA molecule 213 within the agarosegel 207 and 208 and through the nanopore 105.

According to an embodiment, FIG. 3 illustrates multilayers of agarosegels 207, 208, 301 and 302 (e.g., with different concentrations). Theagarose gels 301 and 302 have different pore sizes as compared toagarose gels 207 and 208. The agarose gels 207 and 208 may have the sameconcentration of agarose material.

The agarose gel has different porous structures at differentconcentrations of agarose, and thus provides easy tuning of thepolyelectrolyte confinement and electric field distribution via theagarose gels 207, 208, 301, and 302. The less the concentration ofagarose, the larger the porous structures within the agarose gel. Thehigher the concentration of agarose, the smaller the porous structurewithin the agarose gel.

Assume that the agarose gels 207 and 208 have the same internal porousstructure with pores that may be (about) 100 nanometers (nm) in diameter(and/or smaller), which is based on the concentration of agarosematerial in the agarose gels 207 and 208. Assume that the agarose gel301 has a concentration higher than concentration of the agarose gels207 and 208, and that the agarose gel 301 has an internal porousstructure with pores that may be (about) 250 nm and/or more. Also,assume that the agarose gel 302 has a concentration higher than theconcentration of the agarose gel 301, and that the agarose gel 302 hasan internal porous structure with pores that may be (about) 350 nmand/or more. By having the varied concentrations of agarose from lowestto highest respectively for the agarose gels 207 to 301 to 302, anagarose gel gradient 350 is created as shown in FIG. 3.

If there is a very large DNA molecule 213 with a 2 μm coil diameterand/or greater (coil space) being driven by the electric field towardthe nanopore 105, this large DNA molecule 213 may hit (against) theagarose get 207 but not uncoil when the agarose gel 207 has pores of 100nm (and/or smaller) in diameter. To ensure that the large DNA molecule213 (with the 2 μm coil diameter) uncoils, the agarose gel gradient 350is used. By applying the agarose gel gradient 350, the large DNAmolecule 213 first reaches the agarose gel 302 (with the lowestconcentration of agarose, e.g., (about) 0.5% of agarose material) havingpores 350 nm and/or more in diameter in order to begin uncoiling the DNAmolecule 213. These large pores (350 nm and/or more) in the agarose gel302 start the linearization/stretching of the coiled DNA molecule 213.

Next, the DNA molecule 213 (that has been partially uncoiled) is drivenby the electric field (E) to reach the agarose gel 301 (with the middleconcentration of agarose, e.g., (about) 0.9% of agarose material) havingpores 250 nm and/or more in diameter in order to further uncoil the DNAmolecule 213. These medium sized pores (250 nm and/or more) in theagarose gel 301 further linearize and stretch the DNA molecule 213.

Last, the DNA molecule 213 (that has been further uncoiled) is driven bythe electric field to reach the agarose gel 207 (with the highestconcentration of agarose, e.g., (about) 1.5% of agarose material) havingpores (about) 100 nm (and/or smaller) in diameter in order to completelyuncoil the DNA molecule 213. These small pores (100 nm and/or less) inthe agarose gel 207 linearize and stretch the DNA molecule 213 to fitinto the nanopore 105 completely uncoiled.

Although three grades of agarose gel concentrations and internal poresizes (from largest to smallest) are discussed, some implementations mayhave more than three grades in the agarose gel gradient 350 in order tobegin with even larger internal pores (e.g., 500 nm (and/or more) indiameter) and end with even smaller internal pores (e.g., about 50-75 nmin diameter).

As understood by one skilled in the art, different concentrations ofagarose are formed by mixing more or less water to the agarose material.Agarose is a polysaccharide polymer material. Agarose is available as awhite powder which dissolves in near-boiling water, and forms a gel whenit cools. Agarose gels and melts at different temperatures, and thegelling and melting temperatures vary as understood by one skilled inthe art.

FIG. 4 illustrates a method 400 for reducing entropy of apolyelectrolyte (e.g., molecule 213) before the polyelectrolyte movesthrough the nanopore 105 according to an embodiment. Reference can bemade to FIGS. 1, 2, and 3, along with FIG. 5 discussed below.

The free-standing membrane 100 is provided with the nanopore 105 formedthrough the membrane 100 at block 405. FIG. 1 illustrates shows how tofabricate a single nanopore 105 but it is understood that an array ofnanopores and nanochannels may be formed in the membrane 100.

At block 410, the agarose gels 207 and 208 are formed (poured) ontoeither or both sides of the nanopore 105 in the membrane 100, and theagarose gel 207 and 208 (301, 302) is a porous material.

At block 415, the polyelectrolyte (molecule 213) is uncoiled by drivingthe polyelectrolyte through the porous material of the agarose gel 207(301, 302) via an electric field generated by the voltage of the voltagesource 209.

The electric field drives polyelectrolyte (molecule 213), having beenuncoiled and linearized by the agarose gel, into the nanopore 105 forsequencing. For example, the ionic current can be measured by theammeter 210 as each base of the DNA molecule 213 passes through thenanopore 105, which causes a change in the ionic current thatcorresponds to the identification of the base.

The polyelectrolyte (i.e., molecule 213) is sustained/maintained in anuncoiled and linearized state (i.e., straight) by driving thepolyelectrolyte into the agarose gel 208 on the backside of the membrane100. If the agarose gel 208 were not present on the backside of themembrane 100, the molecule 213 would recoil after exiting the nanopore105 and/or the agarose gel 207 (on the top side), and the molecule 213may begin to recoil (into the coil ball, e.g., shown FIG. 2C) beforeexiting the nanopore 105.

The agarose gel is formed onto both sides of the nanopore 105 in themembrane 100 by applying the agarose gels 207 and 208 respective to atop side and backside of the membrane 100 having the nanopore 105.

The porous material of the agarose gels 207, 208, 301, and 302 havepores of different sizes. The agarose gel may be formed to havedifferent concentrations according to a length of the polyelectrolyte(molecule 213) that is going to be sequence in the nanopore 105. Ashorter length resulting in a smaller coil space may only need oneconcentration of agarose gel 207, while a longer length resulting in alarge coil space needs multiple concentrations of agarose gels 207, 301,and 302. Based on the length of the polyelectrolyte having a coildiameter equal to or greater than a predetermined diameter (e.g., 1×10⁻⁶m in diameter when the long polyelectrolyte is coiled into a ball), theagarose gel gradient 350 is deposited/formed in order to have a highestconcentration of agarose material (e.g., agarose gel 207) closest tonanopore while depositing the agarose gel (e.g., agarose gel 302) with aleast concentration of the agarose material farthest from the nanopore105. The middle concentration of agarose material (agarose gel 301) isin between. For example, the agarose gel is provided onto the top sideof the membrane 100 with a gradient (such as agarose gel gradient 350),such that the gradient of agarose gel has largest pores (e.g., inagarose gel 302) farthest from the nanopore 105 and smallest pores(e.g., in agarose gel 207) closest to the nanopore 105.

The agarose gels 207, 302, and 302 of different concentrationscorrespond to the different internal pore sizes, which are stacked asshown in FIG. 3. The respective pore sizes vary from relatively large atthe top of the stack (gradient) to relatively small at the bottom of thestack near the nanopore entrance in order to allow for fasteraccumulation of the polyelectrolyte near the nanopore entrance ascompared to not having the agarose gel with the different concentrationscorresponding to the different pore sizes in a stack. The pore sizesthat are relatively large may vary/range anywhere from a few microns(such as 1, 2, 3, 4, 5, . . . 7 μm) to sub-100-nanometers (such as 60-90nm) in diameters. The pore sizes that are relatively small correspond toa few tens of nanometers (such as 1, 2, 3, 5, 10, 20, 30 . . . 40 nm) indiameter.

The agarose gel 207 (301, 302) has a different thickness on the top sideof the nanopore as compared to the thickness of the agarose gel 208 onthe backside of the nanopore 105. In one implementation, the agarose gel207 (301, 302) has the same thickness or approximately the samethickness on both the top side and backside (agarose gel 208) of thenanopore 105. When the agarose gel (e.g., agarose gel 207) is placed onone side of the nanopore 105 in the top fluidic reservoir 201, thepolyelectrolyte is put on same side of the nanopore 105 as the agarosegel 207 in the top fluidic reservoir 201. Agarose gel 208 might not beplaced on the backside.

The polyelectrolyte includes single-stranded DNA and double-strandedDNA. Also, the polyelectrolyte includes RNA, peptides, and other chargedlinear polymers.

FIG. 5 illustrates an example of a computer 500 (e.g., as part of thecomputer test setup for testing and analysis) which may implement,control, and/or regulate the voltage of the voltage source 209, andmeasurements of the ammeter 210 and as discussed herein.

Various methods, procedures, modules, flow diagrams, tools,applications, circuits, elements, and techniques discussed herein mayalso incorporate and/or utilize the capabilities of the computer 500.Moreover, capabilities of the computer 500 may be utilized to implementfeatures of exemplary embodiments discussed herein. One or more of thecapabilities of the computer 500 may be utilized to implement, toconnect to, and/or to support any element discussed herein (asunderstood by one skilled in the art) in FIGS. 1-4. For example, thecomputer 500 which may be any type of computing device and/or testequipment (including ammeters, voltage sources, connectors, etc.).Input/output device 570 (having proper software and hardware) ofcomputer 500 may include and/or be coupled to the nanodevices andstructures discussed herein via cables, plugs, wires, electrodes, patchclamps, etc. Also, the communication interface of the input/outputdevices 570 comprises hardware and software for communicating with,operatively connecting to, reading, and/or controlling voltage sources,ammeters, and current traces (e.g., magnitude and time duration ofcurrent), etc., as discussed herein. The user interfaces of theinput/output device 570 may include, e.g., a track ball, mouse, pointingdevice, keyboard, touch screen, etc., for interacting with the computer500, such as inputting information, making selections, independentlycontrolling different voltages sources, and/or displaying, viewing andrecording current traces for each base, molecule, biomolecules, etc.

Generally, in terms of hardware architecture, the computer 500 mayinclude one or more processors 510, computer readable storage memory520, and one or more input and/or output (I/O) devices 570 that arecommunicatively coupled via a local interface (not shown). The localinterface can be, for example but not limited to, one or more buses orother wired or wireless connections, as is known in the art. The localinterface may have additional elements, such as controllers, buffers(caches), drivers, repeaters, and receivers, to enable communications.Further, the local interface may include address, control, and/or dataconnections to enable appropriate communications among theaforementioned components.

The processor 510 is a hardware device for executing software that canbe stored in the memory 520. The processor 510 can be virtually anycustom made or commercially available processor, a central processingunit (CPU), a data signal processor (DSP), or an auxiliary processoramong several processors associated with the computer 500, and theprocessor 510 may be a semiconductor based microprocessor (in the formof a microchip) or a macroprocessor.

The computer readable memory 520 can include any one or combination ofvolatile memory elements (e.g., random access memory (RAM), such asdynamic random access memory (DRAM), static random access memory (SRAM),etc.) and nonvolatile memory elements (e.g., ROM, erasable programmableread only memory (EPROM), electronically erasable programmable read onlymemory (EEPROM), programmable read only memory (PROM), tape, compactdisc read only memory (CD-ROM), disk, diskette, cartridge, cassette orthe like, etc.). Moreover, the memory 520 may incorporate electronic,magnetic, optical, and/or other types of storage media. Note that thememory 520 can have a distributed architecture, where various componentsare situated remote from one another, but can be accessed by theprocessor 510.

The software in the computer readable memory 520 may include one or moreseparate programs, each of which comprises an ordered listing ofexecutable instructions for implementing logical functions. The softwarein the memory 520 includes a suitable operating system (O/S) 550,compiler 540, source code 530, and one or more applications 560 of theexemplary embodiments. As illustrated, the application 560 comprisesnumerous functional components for implementing the features, processes,methods, functions, and operations of the exemplary embodiments.

The operating system 550 may control the execution of other computerprograms, and provides scheduling, input-output control, file and datamanagement, memory management, and communication control and relatedservices.

The application 560 may be a source program, executable program (objectcode), script, or any other entity comprising a set of instructions tobe performed. When a source program, then the program is usuallytranslated via a compiler (such as the compiler 540), assembler,interpreter, or the like, which may or may not be included within thememory 520, so as to operate properly in connection with the O/S 550.Furthermore, the application 560 can be written as (a) an objectoriented programming language, which has classes of data and methods, or(b) a procedure programming language, which has routines, subroutines,and/or functions.

The I/O devices 570 may include input devices (or peripherals) such as,for example but not limited to, a mouse, keyboard, scanner, microphone,camera, etc. Furthermore, the I/O devices 570 may also include outputdevices (or peripherals), for example but not limited to, a printer,display, etc. Finally, the I/O devices 570 may further include devicesthat communicate both inputs and outputs, for instance but not limitedto, a NIC or modulator/demodulator (for accessing remote devices, otherfiles, devices, systems, or a network), a radio frequency (RF) or othertransceiver, a telephonic interface, a bridge, a router, etc. The I/Odevices 570 also include components for communicating over variousnetworks, such as the Internet or an intranet. The I/O devices 570 maybe connected to and/or communicate with the processor 510 utilizingBluetooth connections and cables (via, e.g., Universal Serial Bus (USB)ports, serial ports, parallel ports, FireWire, HDMI (High-DefinitionMultimedia Interface), etc.).

In exemplary embodiments, where the application 560 is implemented inhardware, the application 560 can be implemented with any one or acombination of the following technologies, which are each well known inthe art: a discrete logic circuit(s) having logic gates for implementinglogic functions upon data signals, an application specific integratedcircuit (ASIC) having appropriate combinational logic gates, aprogrammable gate array(s) (PGA), a field programmable gate array(FPGA), etc.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of onemore other features, integers, steps, operations, element components,and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated

The flow diagrams depicted herein are just one example. There may bemany variations to this diagram or the steps (or operations) describedtherein without departing from the spirit of the invention. Forinstance, the steps may be performed in a differing order or steps maybe added, deleted or modified. All of these variations are considered apart of the claimed invention.

While the preferred embodiment to the invention had been described, itwill be understood that those skilled in the art, both now and in thefuture, may make various improvements and enhancements which fall withinthe scope of the claims which follow. These claims should be construedto maintain the proper protection for the invention first described.

What is claimed is:
 1. A system for reducing entropy of apolyelectrolyte before the polyelectrolyte moves through a nanopore, thesystem comprising: a free-standing membrane having the nanopore formedthrough the membrane, the membrane including a silicon substrate, aninsulating layer, and another insulating layer, wherein the insulatinglayer is directly on a top surface of the silicon substrate, wherein theanother insulating layer is directly on a bottom surface of the siliconsubstrate, such that the insulating layer and the another insulatinglayer are not in contact with each other while both the insulating layerand the another insulating layer are in direct contact with oppositesurfaces of the silicon substrate, wherein the nanopore is only formedin the insulating layer and has a uniform diameter in the insulatinglayer such that no nanopore is present in the silicon substrate and theanother insulating layer; a cavity comprising a narrow end and a wideend formed in the silicon substrate, the narrow end being adjacent tothe insulating layer at the nanopore, the narrow end being larger thanthe nanopore; a top fluidic reservoir on one side of the membrane and abottom fluidic reservoir on an opposing side of the membrane, whereinone surface of the insulating layer is positioned directly on thesilicon substrate and an opposite surface of the insulating layer isdirectly exposed to the top fluidic reservoir such that no layer ispositioned on the opposite surface of the insulating layer, wherein onesurface of the another insulating layer is positioned directly on thesilicon substrate and an opposite surface of the another insulatinglayer is directly exposed to the bottom fluidic reservoir such that nolayer is positioned on the opposite surface of the another insulatinglayer; a first agarose gel formed onto both sides of the nanopore in themembrane, wherein the first agarose gel is formed directly on both theinsulating layer and the another insulating layer, the first agarose gelbeing formed directly on sidewalls of the silicon substrate which formthe cavity such that the first agarose gel fills an entirety of thecavity, wherein a top surface of a portion of the insulating layerextends beyond edges of the top fluidic reservoir such that the portionof the top surface of the insulating layer is exposed and free of thefirst agarose gel while the top surface of another portion of theinsulating layer is within the top fluidic reservoir and is in directcontact with the first agarose gel; a second agarose gel formed directlyon the first agarose gel in the top fluidic reservoir, such that thesecond agarose gel and the insulating layer sandwich a portion of thefirst agarose gel in the top fluidic reservoir; a third agarose gelformed directly on top of the second agarose gel in the top fluidicreservoir, the bottom fluidic reservoir being free from the second andthird agarose gels; and an electric field generated by a voltage sourcethat drives the polyelectrolyte through the porous material of theagarose gel to uncoil the polyelectrolyte, wherein driving thepolyelectrolyte, having been uncoiled and linearized by the agarose gel,into the nanopore is for sequencing.
 2. The system of claim 1, whereinthe polyelectrolyte is sustained in an uncoiled and linearized state bydriving the polyelectrolyte into the first agarose gel on a backside ofthe membrane.
 3. The system of claim 1, wherein the first agarose gel isformed onto both sides of the nanopore in the membrane by applying thefirst agarose gel to a top side and backside of the membrane having thenanopore.
 4. The system of claim 1, wherein a porous material of thefirst, second, and third agarose gels have pores of different sizes. 5.The system of claim 1, wherein the first, second, and third agarose gelsare formed to have different concentrations according to a length of thepolyelectrolyte.
 6. The system of claim 1, wherein a wide end of thecavity is formed through the another insulating layer.
 7. The system ofclaim 1, wherein the first agarose gel is formed in the cavity.
 8. Thesystem of claim 1, wherein no intervening layer is present between thenarrow end of the cavity adjacent to the insulating layer at thenanopore.
 9. The system of claim 1, wherein the nanopore is cylindrical.10. The system of claim 1, wherein the nanopore and the cavity areformed in two separate layers.
 11. The system of claim 1, wherein thefree-standing membrane comprises only 3 layers.
 12. The system of claim1, wherein no layer of the free-standing membrane is conductive.